Compositions and methods of using lamellar bodies for modifying linear biological macromolecules

ABSTRACT

Compositions comprising therapeutically effective amounts of lamellar bodies for the modification of linear macromolecules are disclosed. These lamellar compositions are useful in the treatment of conditions or diseases characterized by a preponderance of heavy mucous secretions, such as otitis media, cystic fibrosis, bronchitis, sinusitis and nasal congestion. Methods of treating these diseases and conditions by administering a therapeutically effective amount of a composition to a patient requiring such treatment are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to UK application No.GB0322448.2, filed on Sep. 25, 2003, the disclosure of which isincorporated by reference herein in its entirety. Applicants claim thebenefit of this application under 35 U.S.C. §119 (a-d).

FIELD OF THE INVENTION

The present invention relates to the identification of lamellar bodieswhich modify linear biological macromolecules that may be present inphysiological and pathophysiological secretions and exudates, such asmucus, DNA, actin and bacterial-derived alginate, with respect to theirphysical properties, viscosity, adhesiveness, liquidity andthree-dimensional disposition. The invention further relates tocompositions and methods of treatment using these lamellar bodies.

BACKGROUND OF THE INVENTION

There are a number of occasions and sites where it would be beneficialto modify linear biological macromolecules, such as in disease statesand during surgery. Examples where linear macromolecules are problematicare acute and chronic otitis media and sinusitis, acute and chronicinflammatory disorders of the respiratory tract (including cysticfibrosis), disorders of the alimentary and genito-urinary tracts, andduring surgical procedures on ducts and surfaces of body cavities filledor covered by thickened secretions or exudates. There are nobiologically efficacious substances or methods typically used foralteration or removal of these species of macromolecules, which inpathological situations can result in acute and chronic morbidity andmortality.

On the simple basis of observed viscosity or lubricity, older physiologytexts classified secretions into thick or thin. Thick secretions weredescribed as “mucoid”, whereas thin secretions were considered “serous”in nature. These ill-defined archaic terms are still in use and have notbeen accurately defined and categorized. The term “serous” is applied tothe sparse, thin secretions encountered universally in the large bodycavities, pleura, pericardium, peritoneum, joints and tendon sheaths.This thin secretion is termed “serous” in that, like serum, it does notclot. In the last decade however, serous fluid in body cavities, was forthe first time accurately defined as a secretion of mesothelium in whichphospholipid microbodies were the predominant element in conferring highlubricity and non-stick properties (Dobbie J W, (1988) Ultrastructuralsimilarities between mesothelium and Type II pneumocytes and theirrelevance to phospholipid surfactant production by the peritoneum In:Khanna R, Nolph K D, Prowant B, Eds: Advances in Continuous AmbulatoryPeritoneal Dialysis. University of Toronto Press, Toronto, pp 32-41;Dobbie J W, Pavlina T, Lloyd J, Johnson R C. (1988); Am J Kid Dis.,12:31-36; Dobbie J W, Lloyd J K. (1989), Perit Dial Int, 9:215-221).Subsequently it has been shown that not only mesothelium but many othertissues in respiratory, alimentary and reproductive tracts containvarying densities of “serous” cells, which secrete a similar substance.

Invisible to light and electron microscopy for so long, these findingshave recently been responsible for the concept that the secretory systemfor these phospholipid microbodies constitutes a hitherto unrecognizedmajor biological system throughout the animal kingdom (Dobbie J W.(1996) Perit Dial Int. 16:574-581).

Widespread throughout the animal kingdom, the secretion of mucus frombody surfaces, in association with underlying motile projections of cellsurfaces, cilia, together constitutes an important mechanical system forautomatic clearance of potentially noxious substances. In lung, themucociliary clearance system is the first line of defense againstinhaled particulates, aerosols and pathogens, into its airways. Suchmaterials are adsorbed out of the air stream onto the mucous gelcontained within the airway surface liquid, which coats the ciliatedepithelium. Despite the focus of intensive research in recent years,particularly in relation to the pathogenesis of cystic fibrosis, thereremain unsolved biophysical problems in understanding the function ofmucociliary clearance in all body surfaces. It is with respect to theidentification of compositions for the treatment of diseases andconditions characterized by the preponderance of thick mucous secretionsthat the present application is directed.

SUMMARY OF THE INVENTION

The present invention relates to the discovery that lamellar bodysecretory cells are a biological necessity in sites where thicksecretions such as mucus are produced, and in disease states wherelinear macromolecules possessing significant inherent visco-elastic andadhesive properties might be encountered.

It would be beneficial to provide a substance or method that is able tomodify for therapeutic purposes linear macromolecules with respect totheir physical properties in relation to viscosity, adhesiveness andrheological characteristics.

Accordingly, a first aspect of the present invention provides for acomposition comprising a therapeutically effective amount of lamellarbodies for the modification of linear macromolecules. In a preferredembodiment, the composition is a pharmaceutical composition comprisingthe lamellar bodies and a pharmaceutically acceptable carrier. Inanother preferred embodiment, the pharmaceutical compositions may beused to treat a mammalian subject suffering from a disease or conditioncharacterized by a preponderance of heavy mucous secretions and in needof such therapy. Such conditions include, but are not limited to, OtitisMedia (acute or chronic), Cystic Fibrosis, sinusitis, bronchitis, andnasal congestion. In another preferred embodiment, the subject is ahuman. In another preferred embodiment, the lamellar bodies compriseabout 44-70% phosphatidylcholine, about 15-23% sphingomyelin, about6-10% phosphatidyl ethanolamine, about 2-6% phosphatidyl serine, about2-4% phosphatidyl inositol and about 4-12% cholesterol by weight. Inanother preferred embodiment, the composition further comprises about0-3% by weight of lysophosphatidyl choline. In a further preferredembodiment, the lamellar bodies comprise about 54% phosphatidylcholine,about 19% sphingomyelin, about 8% phosphatidyl ethanolamine, about 4%phosphatidyl serine, about 3% phosphatidyl inositol and about 10%cholesterol by weight. In another preferred embodiment, the compositionfurther comprises about 2% by weight of lysophosphatidyl choline. In yetanother preferred embodiment, the linear macromolecules in need ofmodification are selected from the group consisting of DNA, mucin,actin, and bacterial-derived alginate. These substances may be producedby the host or invading micro-organisms, i.e. they are substancesproduced or released by either prokaryotes or eukaryotes.

A second aspect of the present invention provides for a method oftreatment for acute and chronic otitis media. In a preferred embodiment,the method of treatment comprises the steps of:

-   -   a) inserting a needle through the tympanic membrane;    -   b) introducing a composition including lamellar bodies through        the needle into the ear; and    -   c) allowing the lamellar bodies to modify the viscosity of the        mucin in the ear such that it is capable of draining from the        ear.

In another preferred embodiment, the method of treatment of otitis mediacomprises the steps of:

-   -   a) inserting a needle through the tympanic membrane;    -   b) introducing a composition including lamellar bodies through        the needle into the ear; and    -   c) allowing the lamellar bodies to modify the composition and        the biological properties of the contents present in the        pathological secretion, including linear macromolecules of the        type mucus, DNA, actin and alginate, to the effect that the        physical properties of the secretions such as viscosity and        adhesiveness are altered, permitting therapeutic drainage of the        middle ear.

In another preferred embodiment, the method comprises treating otitismedia where a tympanostomy tube (vent tube) is in place and has becomeblocked by mucus or by an admixture of mucus and DNA, actin, alginate orwax, and comprises the step of introducing a composition includinglamellar bodies into the external auditory canal.

In yet another preferred embodiment, the method of treatment for otitismedia comprises the steps of:

-   -   a) inserting a catheter into the pharyngeal opening of the        Eustachian tube.    -   b) introducing a composition, including lamellar bodies through        the catheter into the ear.

Preferably the composition is introduced into the middle ear.

A third aspect of the present invention provides for a method ofperforming a functional endoscopic sinus surgical (FESS) procedure onthe sinus or sinuses of a patient comprising the steps of:

-   -   a) applying a composition including lamellar bodies to the sinus        or sinuses;    -   b) allowing lamellar bodies in the composition to modify the        physical properties of viscosity and adhesiveness of linear        macromolecules, including mucus, DNA, actin and alginate in the        area of the sinus or sinuses such that the substances are        capable of being removed; and    -   c) introducing a surgical instrument into the nasal passage such        as to remove tissue from the sinus or sinuses.

A fourth aspect of the present invention provides for a method forapplying a composition including lamellar bodies to the respiratorypassages to modify the physical properties of linear macromolecules ofthe type mucus, DNA, actin and alginate in pathologically alteredsecretions. The lamellar bodies modify the viscosity and adhesiveness ofthe secretions, increasing the fluidity of the airways' surface fluidand restoring mucociliary clearance of secretions throughout therespiratory tract.

In a preferred embodiment, the composition containing suspended lamellarbodies is introduced into the respiratory passages as an aerosol spray.In another preferred embodiment, the fluid containing suspended lamellarbodies may be subjected to ultrasonification during administration ofinhaled aerosol. In yet another preferred embodiment, the therapeuticmoieties can be included on or within the phospholipid bilayers whichconstitute the lamellar bodies for more effective access of therapeuticmoieties to sequestered micro-organisms protected by prokaryote- andeukaryote-derived linear macromolecules.

A fifth aspect of the present invention provides for a method forapplying a composition, including lamellar bodies, to diverse parts ofthe body for the symptomatic relief of viscous secretions in patientswith the autosomol recessive disorder, cystic fibrosis, due to the genedefect responsible for the secretion of a protein called Cystic FibrosisTransmembrane Conductance Regulator (CFTR). In a preferred embodiment,the method of symptomatic treatment of the pathological secretions incystic fibrosis is accomplished by applying a composition containingsuspended lamellar bodies which are introduced into the respiratorypassages as an aerosol spray. In another preferred embodiment, the fluidcontaining suspended lamellar bodies may be subjected toultrasonification during administration of inhaled aerosol. In yetanother preferred embodiment, additional therapeutic moieties can beincluded on or within the phospholipid bilayers which constitute thelamellar bodies for more effective access of the therapeutic moieties tosequestered micro-organisms protected by prokaryote- andeukaryote-derived linear macromolecules. Another preferred embodimentprovides for a method of symptomatic treatment of the effects ofblockage of the secretory ducts of vital organs and glands in patientswith cystic fibrosis by applying a composition including lamellar bodiesthrough intermittent or continuous infusion into the drainage system byneedles or appropriate macro- or micro-catheters, or introduction ofslow-release matrices, enteric-coated or biodegradable capsulescontaining lamellar bodies.

Other advantages of the present invention will become apparent from theensuing detailed description taken in conjunction with the followingillustrative drawings.

DETAILED DESCRIPTION OF THE INVENTION

Before the present methods and treatment methodology are described, itis to be understood that this invention is not limited to particularmethods, and experimental conditions described, as such methods andconditions may vary. It is also to be understood that the terminologyused herein is for purposes of describing particular embodiments only,and is not intended to be limiting, since the scope of the presentinvention will be limited only in the appended claims.

As used in this specification and the appended claims, the singularforms “a”, “an”, and “the” include plural references unless the contextclearly dictates otherwise. Thus, for example, references to “themethod” include one or more methods, and/or steps of the type describedherein and/or which will become apparent to those persons skilled in theart upon reading this disclosure and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the invention, the preferred methods andmaterials are now described. All publications mentioned herein areincorporated herein by reference in their entirety.

Definitions

“Lamellar bodies or microbodies” as used throughout this document,refers to phospholipid, multilamellar, bilayered structures present inmany tissues throughout the body, but also refers to the syntheticmultilayered phospholipid structures having the novel compositiondescribed in the present invention. Thus, this term refers to bothnaturally occurring and synthetic lamellar bodies.

“Lamellasomes” as used herein refers to synthetically prepared lamellarbodies as described by the methods of the present invention.

“Serous” as used herein refers to an exudate or effusion that is thinand watery, and which lacks a significant cellular component.

“Treat”, Treating” or “Treatment” refers to therapy, prevention andprophylaxis and particularly refers to the administration of medicine orthe performance of medical procedures with respect to a patient, foreither prophylaxis (prevention) or to cure or reduce the extent of orlikelihood of occurrence of the infirmity or malady or condition orevent in the instance where the patient is afflicted.

A “therapeutically effective amount” is an amount sufficient to decreaseor prevent the symptoms associated with the conditions or deficienciescontemplated for therapy with the compositions of the present invention.

“Slow release formulation” refers to a formulation designed to release atherapeutically effective amount of a drug or other active agent such asa polypeptide or a synthetic compound over an extended period of time,with the result being a reduction in the number of treatments necessaryto achieve the desired therapeutic effect. In the matter of the presentinvention, a slow release formulation would decrease the number oftreatments necessary to achieve the desired effect.

“Combination therapy” refers to the use of the agents of the presentinvention with other active agents or treatment modalities, in themanner of the present invention for treatment of otitis media orabdominal surgery. These other agents or treatments may include drugssuch as corticosteroids, non-steroidal anti-inflammatory compounds, orother agents useful in treating or alleviating pain. The combined use ofthe agents of the present invention with these other therapies ortreatment modalities may be concurrent, or the two treatments may bedivided up such that the agent of the present invention may be givenprior to or after the other therapy or treatment modality.

“Local administration” means direct administration by a non-systemicroute at or in the vicinity of the site of an affliction, disorder, orperceived pain.

“Modify” or “modification of” means to alter the physical status of amaterial with respect to viscosity, adhesiveness and/or fluidity. In thepresent invention “modification” refers to changes in physicalcharacteristics, perhaps due to changes in hydration of the gel,although other factors may play a role in modification of the physicalstate.

“Acute otitis media” is an inflammation of the area behind the eardrum(tympanic membrane) in the chamber called the middle ear. Acute otitismedia is an infection that produces pus, fluid, and inflammation withinthe middle ear. Acute otitis media frequently occurs as an aftereffectof respiratory infections as the nasal membranes and eustachian tubebecome swollen and congested.

“Chronic otitis media” refers to a middle ear infection that may developwhen infection in the middle ear persists for more than 2 weeks. Themiddle ear and eardrum may start to sustain ongoing damage occasionallyresulting in drainage through a nonhealing hole in the eardrum.

Throughout this document the term “linear macromolecule” refers tomolecules which are linear at the molecular level and whose structureessentially comprises the multiple repetition in linear sequence ofunits derived from molecules of low relative molecular mass. Themolecules may possess side-chains, but these will not typicallyparticipate in chemical cross-linking. Examples of linear macromoleculesof this species include mucus, DNA, actin and alginate.

“Therapeutic moieties” refers to any therapeutically effective molecule,whether it is a small organic chemical compound, or a protein orpeptide, or a nucleic acid, or an antibody or antibody fragment, or acarbohydrate, that may be attached to the lamellar bodies andadministered to subjects suffering from diseases or conditions for whichtreatment may be beneficial. Optionally, therapeutic moieties can beincluded on or within the phospholipid bilayers which constitute thelamellar bodies.

General Description

It was not until the time of the present invention that treatment ofdiseases having thick mucoid secretions with compositions containing thelamellar bodies of the present invention was contemplated. That is, arecent key advance in molecular biology and physiology has been therealization that the thin “serous” secretions which have high lubricityand do not form gels (clots), can exist on their own in body cavities,ducts, glands, surfaces, etc, whereas thick, “mucoid” secretions are innormal physiological states always accompanied by cells which secrete aserous solution containing phospholipid microbodies.

The invention described herein has provided new observations andexplanatory concepts resulting in the conclusion that a biological agentwhich counteracts the viscosity of thick mucous secretions is alwaysprovided in the form of accompanying “serous” secretory cells. Thelikeliest function of phospholipid microbodies on gels consisting oftangled linear polymers would be the modification of their adhesive andvisco-elastic properties.

Studies on the effect of synthetic lamellar bodies on the adhesiveness,viscosity and fluidity properties of mucous secretions has for the firsttime provided observations that the key constituents of “serous”secreting cells (lamellar bodies) significantly modify the physicalproperties of mucus. It has further been shown that synthetic lamellarbodies also have a significant effect on the physical properties ofpathophysiological thick secretions, which contain polymeric DNA, actinor alginate derived from dead host cells and bacteria.

Therapeutic Uses and Compositions

Chronic Otitis Media With Effusion (COME)

COME is an extremely common condition, particularly in children.Following an acute or repeated acute attacks of otitis media, the middleear may become filled with a viscous secretion referred to as “glueear”. This can become an intractable problem with serious consequencesfor hearing and in many cases, delayed or impaired speech and learning.Venting through grommets is the mainstay of present-day practice. Thereis widespread agreement among otolaryngologists that current methods oftreatment are often sub-optimal in their outcome. Although much recenteffort has been deployed into research to find better modes oftreatment, it is unfortunate that only minimal attention has been paidto the little known finding of serous secreting cells in the Eustachiantube and middle ear.

Nature of the Epithelial Lining of the Middle Ear and the EustachianTube

The nature of the lining of the Eustachian tube and middle ear has forlong been considered to be a “modified” respiratory epitheliumcontaining both ciliated and goblet cells derived from the basal layers.However, based on investigations of whole mounted histologicalpreparations from fetuses, premature infants, infants, normal childrenand adults, Tos concluded that mucous secreting cells are not a normalbut a pathological component of middle ear epithelium (Tos M,Caye-Thomasen P, (2002), J Oto-Rhino-Laryngology & Related Specialties.64:86-94). Indeed, it is now increasingly accepted that the liningepithelium throughout the Eustachian tube and middle ear in normalhealth, secretes but a sparse, thin solution which merely confers aglistening appearance to the membrane. The only cells likely to beresponsible are loosely described in the literature as “serous” cells.

Aetiopathogenesis of Chronic Otitis Media With Effusion (COME)

It is now accepted that the key factor in the aetiopathogenesis of COMEis the metaplastic transformation of middle ear epithelium to apredominantly goblet cell layer engaged in long-term hypersecretion ofmucus. Recent research into the pathophysiology of COME has demonstrateda continuous chain of reactions precipitated by bacterial infection,where stimulation by endotoxins, together with pro-inflammatorycytokines (IL1β, TNFα, IL8, PAF) and growth factors (ErbB & HepGF) arenot only powerful promoters of goblet cell hyperplasia, but also act ashighly potent secretogogues of mucus.

Functions of Lamellar Body Secretory System

In pulmonary alveoli the prime function of lamellar bodies is theprovision of surfactant phospholipids to promote gaseous exchange at thefluid-air interface, and to lower the opening pressure of alveolar wallsduring inspiration.

In mesothelial-lined cavities, we have shown the function of lamellarbodies to be that of lubrication and provision of a non-stick surface.This is achieved through the formation of microscopic, multilamellarball and roller bearings which constantly form and reform betweensliding surfaces.

In open body surfaces and ducts, upper respiratory, gastro-intestinaland reproductive tracts, we have produced evidence that, in addition toconferring non-stick properties, a key function of lamellar bodysecretion in these sites is the modification and/or disaggregation oflocally produced gels, such as mucus. This extremely important propertyis the biological guarantee that vital surfaces, ducts and tubes have ahighly conserved method for preventing smothering and blockage byviscous secretions.

Lamellar Body Secretion in Middle Ear

In 1984 it was first demonstrated that the epithelial lining of the ratEustachian tube synthesised phosphatidylcholine, the principalconstituent of pulmonary surfactant, in amounts comparable to thatproduced by pulmonary alveolar epithelium (Wheeler S L, Pool G L, Lumb RH. (1984), Biochim Biophys Acta, 794:348-349). This was the firstindication that the Eustachian tube epithelium produced a secretion withsurfactant properties. Although this paper stimulated Dobbie et al(Dobbie J W, Pavlina T, Lloyd J, Johnson R C, (1988), Am J Kid Dis,12:31-36) to apply their methodology to mesothelium, Wheeler'spioneering observations failed to evoke much interest or research in theotolaryngological community. In the last several years however, a smallnumber of research papers on the ultrastructure of middle ear epitheliumhas confirmed by electron microscopy the presence of lamellar bodysecreting cells, so-called “serous” cells (Mira E, Bonazzo M, Galioto Pet al. (1988), J Autorhino Laryngology & Related Specialities,50:251-256.).

New Concepts on the Function of Phospholipid Secretion in Normal Stateand Pathological Conditions in Middle Ear

A review of recent research carried out on the epithelial lining of themiddle ear, considered in relation to our comparative ultrastructuraland physiological research on lamellar body secretion at all othernon-pulmonary locations, indicates that in the normal state, a constantthin secretion of lamellar bodies has a prime function in providing anon-stick surface in the Eustachian tube, tympanic cavity and air sacs,as we have demonstrated in the mesothelial-lined cavities of the body.In this site formation of adhesions, synechiae or outright blockage ofthe ductal portions of the system must be avoided at all costs. Thus itwould have been surprising indeed if the benefit of highly conservednon-stick properties of lamellar bodies had not been present in theseimportant cavities.

Like the upper respiratory tree, the middle ear, under the stimulus ofinflammation, has the ability to produce mucous secreting goblet cells.However, current understanding of the physiology and pathophysiology ofmiddle ear has not yet become aware of the dual, balanced system ofmucus and lamellar body secretion.

The preparation of synthetic lamellar bodies now allows, for the firsttime, an opportunity to examine the interaction in a controlled ex vivomodel, the possible physico-chemical interaction between phospholipidmicrobodies and naturally-occurring gels consisting of tangled networksof linear glycoprotein polymers.

“Surfactant” In The Middle Ear

In the last several years there has been a quickening of interest in thepossible role of “surfactant” in the physiology and pathophysiology ofthe middle ear. The theme of the investigations has assumed thatsurfactants are in some way involved with ventilation, protection andclearance of the middle ear through the Eustachian tube. These functionshave been examined by administration of exogenous surfactant and havesuccessfully demonstrated that surfactant decreased the opening forcesof the Eustachian tube, even in otologically healthy rats (Van HeerbeekN, Tonnaer E L G M, Koen J A O et al. (2003), Otology & Neurotology, 24:6-10). In this capacity however, the lamellar bodies supplied in thesurfactant are not functioning as a surfactant as such, but as acoating, which prevents adhesion of opposing surfaces. No significanteffect however was observed on mucociliary clearance. In this respectthere is an on-going failure to recognize that it is now establishedthat the lubricating and non-stick properties of lamellar bodiesconstitute the major function of this system, and its pulmonarysurfactant properties are a late, highly specialized evolutionarydevelopment. Similarly, it has not yet dawned on workers in this fieldthat it would be a worthwhile experiment to mix these lubricatingmicrobodies with gels of linear macromolecules such as mucus and DNA,which are pivotal in middle ear pathology.

This is not to deny the importance of the lamellar bodies' role as asurfactant in the gaseous exchange which occurs across the middle earand associated air cells, and must play some significant part inventilation. This well-known function of surfactant however, hasblind-sighted researchers from investigating the more mundane role oflamellar bodies as a “biological lubricant and cleanser” of thicksecretions.

Secretion of Lamellar Bodies by Serous Cells in the Mucosa of the NasalCavities and Sinuses

It has recently been shown that there are scattered throughout thesecavities serous cells which have been shown by electron microscopy tosecrete typical lamellar bodies. In chronic infection, under thestimulus of endotoxins, exotoxins, and pro-inflammatory cytokines, thereis a hyperplasia of goblet cells leading to excessive mucous secretion.These cavities are then liable to blockage and interruption of drainagedue to thick secretions of viscous mucus, DNA, actin andbacterial-derived alginate.

Therapeutic application of lamellar bodies by aerosol spray typicallywill be used in disorders of the respiratory passages in whichpathophysiological changes in the consistency of secretions due toalterations in concentration and/or physical status of linearmacromolecules are acute and chronic organismal-induced inflammation ofthe respiratory passages (acute and chronic bronchitis, bronchiectasisetc), acute and chronic inflammation induced by allergens (organic andinorganic) as in all types of asthma. Increased viscosity of secretionscaused by inhalation of irritant or toxic particulate inducingalteration in the thickness of airway secretions as in for example,byssinosis due to inhalation of cotton dust and bagassosis, inhalationof bagasse (dried sugar cane refuse). The application of lamellar bodieswill also be made to the respiratory passages to disperse extracellularpolymeric substance (EPS), a protective biofilm secreted by diversepathogenic micro-organisms, including Pseudomonas aeruginosa, in chronicrespiratory disorders.

Use of Lamellar Bodies to Treat Cystic Fibrosis

The present invention also provides for a method for applying acomposition, including lamellar bodies, to diverse parts of the body forthe symptomatic relief of viscous secretions in patients with theautosomol recessive disorder, cystic fibrosis, due to the gene defectresponsible for the secretion of a protein called Cystic FibrosisTransmembrane Conductance Regulator (CFTR). This renders the cellmembrane permeant to chloride and other larger organic anions. Thecondition is characterized by gross increases in the concentration andviscosity of mucus in the respiratory, alimentary, gastro-intestinal andreproductive tracts. Chronic, high density colonization of secretions bybacteria, leads to sequestration of high concentrations of polymeric DNAand actin from dead host leucocytes and bacteria which further thickenthe secretions, paralyzing mucociliary clearance. Also, colonization byorganisms which actively secrete protective biofilms includingPseudomonas aeruginosa which produce a viscous linear macromolecule(alginate), further thickening pathological secretions and creatingphysical barriers, rendering the organism inaccessible tochemotherapeutic attack. In cystic fibrosis, respiratory disease is theprincipal cause of mortality.

There are no biologically efficacious substances or methods typicallyused for modifying the physical properties these species of linearmacromolecules which, in concert, are responsible for dysfunctionalsecretions in cystic fibrosis.

The present invention provides for a method of symptomatic treatment ofthe pathological secretions in cystic fibrosis by applying a compositioncontaining suspended lamellar bodies which are introduced into therespiratory passages as an aerosol spray. Optionally, the fluidcontaining suspended lamellar bodies may be subjected toultrasonification during administration of inhaled aerosol.

Optionally, therapeutic moieties can be included on or within thephospholipid bilayers which constitute the lamellar bodies for moreeffective access of therapeutic moieties to attack sequesteredmicro-organisms protected by prokaryote- and eukaryote-derived linearmacromolecules.

Although pathological secretions in the respiratory tract are theprincipal cause of morbidity and mortality, their effect on othersurfaces, passages and ducts results in significant lesions due toblockage and cyst formation particularly in the alimentary andgenito-urinary tracts in patients with CF. As for the respiratory tract,there are no biologically efficacious substances or methods typicallyused in cystic fibrosis for modifying the effects of linearmacromolecules in extrapulmonary sites.

The present invention also provides for a method of symptomatictreatment of the effects of blockage of the secretory ducts of vitalorgans and glands in patients with cystic fibrosis by applying acomposition including lamellar bodies through intermittent or continuousinfusion into the drainage system by needles or appropriate macro- ormicro-catheters, or introduction of slow-release matrices,enteric-coated or biodegradable capsules containing lamellar bodies.

This method of symptomatic treatment is appropriate for ductalstructures and passages throughout the entire body, including thesurfaces of the gastro-intestinal tract with particular relevance to thethickened membrane covering the mucosa of the small bowel, pancreaticand hepato-biliary drainage network, the genito-urinary system,including intra-renal tubules, medullary drainage system, renal pelvis,ureters, bladder, bladder diverticula, urethra, vesico-colic fistula,ileo-vesicle fistula, vas deferens, seminal vesicles, prostate, cervix,endometrium, Fallopian tube mucosa, salivary and lachrymal glands.

Optionally, therapeutic moieties can be included on or within thephospholipid bilayers which constitute the lamellar bodies for moreeffective access of therapeutic moieties to sequestered micro-organismsprotected by prokaryote- and eukaryote-derived linear macromolecules.

Basic laboratory testing of the effect of lamellar bodies (with thetesting being carried our using synthetic lamellar bodies) on theadhesiveness, viscosity and fluidity properties of secretions has, forthe first time, shown that the lamellar bodies significantly modify thephysical properties of mucus. It has further been shown that syntheticlamellar bodies also have a significant effect on polymeric DNA andactin which can be derived from dead host cells and bacteria. Likewise,synthetic lamellar bodies have been shown to modify physical properties,including viscosity and adhesiveness of alginate of the type secreted bypathogens responsible for a range of life-threatening disorders.

Physical and Biological Properties of Mucous Secretions

Mucus is the archetypal slimy substance, which coats many epithelialsurfaces and is secreted into fluids, such as airway surface fluid,saliva and gastro-intestinal juices. Mucus forms a layer adherent toepithelial surfaces of the respiratory, alimentary and genito-urinarytracts. There it acts to entrap micro-organisms, to provide a diffusionbarrier against contact with noxious substances (e.g. inhaled smoke,gastric acid) and allegedly to serve as a lubricant to minimize shearstress damage to the delicate membranes.

Mucus is composed of polymeric glycoproteins suspended in water thatcontains electrolytes. It has an extended polypeptide backbone(apomucin), with numerous oligosaccharide side chains. Covered byabundant polyanionic charges, mucus, on exocytotic release from gobletcells, becomes extensively hydrated. Resistant to dehydration, itpossesses unique rheological properties of high elasticity, adhesivenessand low solubility. Mucin monomers are polymerized through end-to-enddisulphide bonds to form linear polymers which in respiratory mucus areat least 30μ long. Mucus forms a gel whose three-dimensional structurecomprises a tangled network of linear mucin polymers devoid ofintermolecular cross bridges. In airway surface liquid in normalindividuals, mucin concentration is 1% by weight. In cystic fibrosis, aninherited respiratory disorder, mucin concentration is 3-4%.

Widespread throughout the animal kingdom, the secretion of mucus frombody surfaces, in association with underlying motile projections of cellsurfaces, cilia, together constitutes an important mechanical system forautomatic clearance of potentially noxious substances. In lung, themucociliary clearance system is the first line of defence againstinhaled particulates, aerosols and pathogens, into its airways. Suchmaterials are adsorbed out of the air stream onto the mucous gelcontained within the airway surface liquid that coats the ciliatedepithelium. The focus of intensive research in recent years,particularly in relation to pathogenesis of cystic fibrosis, thereremain unsolved biophysical problems understanding the function ofmucociliary clearance in all body surfaces.

Research work on mucus carried out by the inventor has revealed totallynew insights into the functioning of mucus throughout the body. Dataprovided by these studies supports the hypothesis that where themucociliary system is present it is invariably accompanied by anintegrated parallel system whose secretory product modifies and controlsthe physico-chemical, and biological properties of mucus. Through theunmasking of the hitherto unrecognized nature and importance ofrespiratory serous secretions as an essential part of the lamellar bodysecretory system, satisfactory explanations for anomalous observationsand unsolved problems of the physics, molecular biology andpathophysiology of mucociliary clearance have been found.

Until now, mucous secretion has been viewed as a coherent,self-contained secretory system whose principal function is to provide asubstance, which serves as the major protectant of epithelial surfacesthroughout the body. Although recent research has demonstrated that thesecretion of mucus by goblet cells is responsive to a very wide range offactors from organismal products, through cytokines to neural andhormonal stimuli, there has been no appreciation that a parallel systemof secretory cells releases microbodies onto epithelial surfaces whichhave a profound modifying effect on the consistency, properties anddisposal of mucus. This is the much neglected and poorly understoodsecretion of the serous cells that are always found in association withgoblet cells.

It has long been recognized that throughout respiratory airways therewere two cell populations in the surface epithelium, the predominanttype being mucous secreting cells called goblet cells and the lesserknown serous secreting. Whereas goblet cells release a thick or viscoussecretion, serous cells were credited with the production of a thinwatery solution. Mucous secretion has received by far the most attentionfrom biological researchers, in comparison to the serous cells whosesecretion has been largely ignored. The reason for the disregard lies inthe unfortunate failure to identify accurately the nature of thesecretion. This was due to the fact that tissue from the respiratorypassages, fixed and processed using standard techniques for examinationby electron microscopy, did not preserve the contents of the secretoryvesicles within the serous cells which appeared to be empty. Whenhowever, the cells were fixed with tannic acid and glutaraldehyde, thevesicles are shown to contain osmiophilic phospholipid bilayers arrangedin complex geometric patterns. These are lamellar bodies.

Of the few investigations carried out on serous cells, it was observedthat those in the respiratory tree, sometimes called Clara cells, didproduce phospholipid microbodies. However, the possibility that airwayssurface fluid contained a balanced mixture of thick, visco-elasticpolymer and a highly efficient lubricant microbody has not beenconsidered by research workers in this field. Indeed, the inventor hasdemonstrated that this dual partnership of mucus and lamellar bodieslocally produces a combined and balanced solution with two ingredientswith opposing physico-chemical properties. The body surfaces in therespiratory, gastro-intestinal and genito-urinary tract contain cellsthat secrete mucus and cells that secrete lamellar bodies. The relativeproportions of each type of cell varies from site to site according tothe proximity to major, local changes in anatomy, e.g. sphincters, andin time and rate of secretion in response to physiological orpathological stimuli.

In conclusion, the recent discoveries of the inventor, using electronmicroscopy, of the ubiquity of lamellar body secreting cells andespecially the demonstration that they are found in tandem with mucoussecreting cells, has provided new insights into the existence of abalanced system which exercises control over the micro-environment ofsurfaces and ducts. As predicted by ultrastructural research on thisrecently discovered secretory system, the creation of synthetic lamellarbodies has provided for the first time in vitro confirmation of themodifying effect of lamellar bodies on the physical properties ofsecretions and exudates containing linear macromolecules.

DNA, Actin And Alginate In Pathological Secretions And Exudates

All of these substances are recognized as viscous molecules released inconsiderable amounts in a range of pathological processes, principallybut not exclusively associated with organismal-induced inflammation.They may be involved usually with mucus, individually or in concert, ina conglomerate thickening of secretions or exudates in a variety ofdisorders, resulting in serious organ and tissue dysfunction throughblockage of ducts and passages, including the Eustachian tube and middleear in chronic otitis media, and in forming impenetrable layers onworking surfaces.

There would appear to be a provision throughout the animal kingdom forthe modification of the physical properties of these linear biologicalmacromolecules.

DNA and Actin

DNA released from the nuclei of dead cells forms an extremely stickysubstance. At a concentration of only 1%, polymeric DNA forms a solidgel in vitro. The volume of DNA released from dead leucocytes in aninflammatory process is considerable. Thus, sputum in cystic fibrosismay contain up to 10% DNA by weight. The linear macromolecule actin,principal constituent of the filamentous cytoskeleton, constitutes 20%of the total protein present in the average cell. This substance,likewise, is a viscous molecule which is released in significant amountsby dead leucocytes, further adding to the viscosity of inflammatorysecretions and exudates. Additionally, both DNA and actin are releasedin not inconsiderable amounts from dead bacteria when present ininfective processes.

Bacterial-Derived Alginate

The bacterium, Pseudomonas aeruginosa, causes serious chronic infectionsthroughout the body, including chronic otitis media, chronic sinusitis,and especially in the respiratory tract in patients with cysticfibrosis. Mucoid colonies of these organisms secrete an extracellularpolymeric substance (EPS) known as exopolysaccharide alginate whichsignificantly enhances their pathogenicity. The alginate is astraight-chain, hydrophilic, colloidal, polyuronic acid composedprimarily of anhydro-beta-D mannuronic acid residues with 1-4 linkage. Aviscous linear macromolecule, this alginate not only contributessignificantly to the thickening of secretions colonized by the bacteria,but forms a biofilm which protects it from phagocytosis by macrophages.

These linear macromolecules constitute a serious hazard to drainage andclearance of secretions and exudates.

Surgical Procedures Where Mucus, DNA, Actin and Alginate are a Problem

In many disease states the overproduction of mucus often occurs and isparticularly problematic when surgical procedures are to be carried out.In most sites and conditions the overproduction of mucus is accompaniedby pathological production and release of linear macromolecules (DNA,actin, alginate). Even in cases where mucus production is normal it canstill be obstructive in surgical procedures.

Functional Endoscopic Sinus Surgery (FESS)

FESS is a minimally invasive procedure for the treatment of chronicsinusitis. It is designed to remove blockage and provide free drainageof the sinus. FESS causes little tissue damage and there is no visiblescarring that is associated with open surgery. FESS is often recommendedin cases of chronic sinusitis that do not respond well to medication orother forms of treatment. This occurs when the sinuses are blocked andunable to drain the mucus. When this happens, the sinuses can becomeinfected.

The FESS procedure uses an endoscope, which enables the surgeon toidentify and correct anatomic causes of sinus outflow obstruction. Othersurgical instruments may be inserted alongside the endoscope to treatproblems inside the sinuses. The kind of surgery will depend on theextent of the sinus problem. Sometimes other nasal procedures may beperformed with the sinus surgery to improve access and drainage of thesinuses. In the present case we are suggesting that lamellar bodies orsynthetic lamellar bodies can be used to modify the mucus prior to orduring the procedure.

Typically the procedure will include the steps of:

-   -   applying a composition including lamellar bodies to the sinus    -   allowing the lamellar bodies in the composition to modify the        viscosity of the mucus and any admixed linear macromolecules        such as DNA, actin or alginate in the area of the sinus such        that it is capable of being removed    -   introducing a surgical instrument to the nasal passage such as        to remove tissues from the sinus

The amount of lamellar bodies in the composition is variable buttypically the dosage concentration of lamellar bodies in the compositionwill be 10×10⁹ per ml. The composition can take the form of a spray(nebulized, with and without ultrasonification), an injection or aslow-release preparation.

Other Surgical Areas

As well as being useful in ear, nose and throat procedures such as FESSand surgical treatment of Otitis Media, lamellar bodies can also be usedin tract and ductal surgery where the secretion of mucus with or withoutadmixtures of linear macromolecules such as DNA, actin and alginate, canmake the procedure more difficult. Most commonly this will be applicablein surgery of the hepato-biliary ducts and gall bladder, pancreaticsurgery, liver surgery, tracheal surgery, reproductive tract surgery,surgery of the salivary and lachrymal glands, including flushing ofducts, and surgery or descaling of the gastro-intestinal tract (in thecases the term surgery relates to procedures that will typically becarried out by a surgeon).

Fistula and Pathological Sinuses

“Fistula” is a term which describes the formation of an abnormalpassageway which develops due to a pathological process. A fistula isusually a narrow tube lined not with an epithelial surface but withgranulation tissue, which connects two body surfaces, either between twohollow viscera or between a hollow viscera and a body surface. Examplesof this are a fistula which develops between colon and urinary bladder(vesico-colic fistula), and vagina and colon (vagino-colic fistula).Such lesions are notoriously difficult to eradicate and give rise topersistent focal, chronic and acute-on-chronic inflammation.Intermittent blockage and/or loculation of the fistula's lumen bythickened secretions of varying admixtures of mucus, DNA, actin andalginate are the key players in its maintenance and create seriousobstacles to its surgical removal and closure. Instillation of topicalantibiotics has minimal effect, due to blockage and coating of thefistula wall by thickened, impenetrable secretions. What is currentlyunavailable to surgeons is a biologically effective substance which willcleanse the fistula of the secretions harboring bacteria, endotoxin,pro-inflammatory cytokines and provide a period of continuous drainageto allow suppression of the inflammation in the wall of the fistula andclearance of pathogenic bacteria to permit successful surgical excisionof the fistula's tract. Fistula are typically encountered in chronicinflammatory bowel disorders, including Crohn's disease and ulcerativecolitis.

In the present case it is suggested that lamellar bodies or syntheticlamellar bodies can be used as a preparatory treatment as an adjunct forsuccessful surgical treatment or eradication of pathological sinuses andfistulae. Typically, the procedure will include endoscopy of theaffected viscera or organ to gain access for inserting a catheter intothe sinus tract for applying a composition, including lamellar bodies,thereby introducing a flushing solution containing lamellar bodies orintroduction of slow-release matrices, enteric-coated or biodegradablecapsules containing lamellar bodies.

Other Treatment Areas Where Modification of Mucus is Therapeutic

In a wide range of disorders, secretions may become so thickened that,rather than protecting surfaces or ducts, their viscosity causes severelocal functional derangement. The pathophysiology responsible for thealteration in physical properties of the secretions is only partiallyunderstood. A prime cause of thickened mucous secretion is stimulationby endotoxins and cytokines, resulting in a proliferation of cells thatsecrete mucus. It is also recognized that the degree of hydration ofmucus has a significant effect on its viscosity (particularly in casessuch as cystic fibrosis).

Radiation-Induced Mucositis

Following radiation therapy of the head and neck, areas of thenasopharynx, pharynx, larynx and trachea may exhibit a troublesomeinflammation of the epithelial surfaces, resulting in thick exudates andsecretions. These viscous, adhesive membranes harbour bacteria,endotoxins and pro-inflammatory cytokines which promote and prolongchronic inflammation at these sites, preventing healing. There isprovided a method for applying a composition including lamellar bodiesin a variety of forms including aerosol spray with or withoutultrasonification, a suspension or gel to the areas affected bymucositis for the dispersement of the adhesive membrane, promotinghealing of the inflamed areas.

Sinusitis

Sinuses are membrane-lined openings in the bones around the nose. Fourpairs of sinuses are connected to the nasal cavity by small openings andthese sinus cavities rest alongside the nose, above the eyebrows andbehind the cheekbones.

Normally, air passes in and out of the sinuses and mucus drains from thesinuses into the nose. When there is an obstruction, fluid is unable topass through the sinuses freely. When sinuses are infected, membranesbecome swollen as blood rushes to the infected area. This swellingcauses congestion, facial pain and increased mucus production. As aresult, the nose may become runny and drippy. The mucous secretions maythicken over time creating a breeding ground for infections.

Acute sinusitis typically follows a cold and lasts for a few weeks. Inthe case of chronic sinusitis, the sinusitis may recur a number of timesover the year. Typically, antibiotics and decongestants are used totreat the condition, although in the case of chronic sinusitisadditional treatment methods are also often used.

In the present invention, it is suggested that a composition containinglamellar bodies can be used to treat sinusitis. The composition couldtake any form but typically would be in the form of a spray or nebulizedformulation. As lamellar bodies modulate the viscosity of mucus thiswould help to relieve the symptoms of sinusitus. It can also be seenthat a similar or the same formulation could be used to treat symptomsof the common cold or influenza. This would be particularly useful inthe form of a nasal spray.

Cystic Fibrosis

Cystic fibrosis (CF) is the most common, lethal, autosomal recessivedisorder in people of European extraction, affecting one in fourthousand infants born per annum in the United States and Canada.Patients with CF die at an early age of respiratory failure due toprofound lung injury secondary to colonization by pathogens and chronicinflammation of pulmonary tissues. The median survival age in NorthAmerica for patients with CF had by 1996 reached thirty, whereas inSouth America it was nine years. Thus socio-economic conditions andavailability of resources determine whether or not patients receiveoptimal care. These factors play an important role in the morbidity andlongevity of individuals with this disorder.

The genetics of CF are now relatively well-understood. As an autosomalrecessive disorder, both parents of an affected infant must carry thegene, and each pregnancy carries a one in four chance of producing achild with CF. The carrier rate in Europeans and their descendants isabout one in twenty-eight, with the rate in other ethnic groupsgenerally being lower. In 1989 the gene responsible for CF was locatedon the long arm of chromosome 7. Mutations cause either deficiency orabsence of a membrane-spanning glycoprotein responsible for themaintenance of the ion channel which allows efflux of anions through thecell membrane in epithelium throughout the body. The protein is calledthe Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). Thischannel renders the cell membrane permeant to chloride and other largerorganic anions such as reduced glutathione (GSH). Despite thisknowledge, there is however, a missing linkage between the genetics andthe resultant pathology in CF.

The CFTR gene is found in epithelial cells of the respiratory,intestinal, hepato-biliary and reproductive tracts. It is also found inexocrine glands such as the serous glands in lung and in pancreas.Absence or dysfunction of the CFTR protein results in thick, viscous,mucous secretions in sinuses, lungs, biliary cannaliculae, intestinesand epididymis, resulting in bronchiectasis, chronic liver disease,biliary and intestinal obstructions, infertility, pancreaticinsufficiency and diabetes.

The essential pathological features of CF can be explained throughblockage by viscous secretions of the cannaliculae, ducts and passagesin tissues and organs where the lining epithelia are affected by thegenetic deficiency. This results in progressive cyst formation, chronicinflammation and infection which weakens then destroys the walls of theducts and passages, typically as encountered in bronchiectasis andpulmonary fibrosis, which are responsible for the greatest threat tolife. Nevertheless, current research acknowledges the existence of amissing linkage between the genetics, its molecular expression and thepathogenesis of the lesions in the different tissues which characterizesCF as a defined disorder.

When it was discovered that the CF gene was responsible for the CFTRprotein which controlled a chloride channel, its role in controlling thevolume and electrolyte composition of sweat was established. Thus it wasnaturally assumed that a similar dysfunction in lung could explain theincreased viscosity of the mucus via the hypothesis of abnormal saltconcentration and low volume of airway surface fluid. Unfortunately,most evidence to date indicates that airway surface fluid is isotonicand of normal volume. Since electrolytes together with colloids are thekey determinants of water distribution in tissue spaces, it was realizedthat the effect of the CFTR protein must be responsible for deficienciesother than electrolytes in the micro-environment within ducts and onsurrounding epithelial surfaces. Therefore, in a continuing search for amolecular linkage between the gene defect and the resultant pathology,deficiencies of other anions have been sought. Of these, the ubiquitoustripeptide, reduced glutathione (GSH), one of the body's most importantwater-soluble anti-oxidants, has been considered a candidate for themissing linkage. GSH is present in extracellular epithelial fluids suchas the airway surface fluid, gastro-intestinal juices, semen, etc. Inthe extracellular milieu, GSH provides powerful anti-oxidant protectionto surfaces heavily exposed to reactive oxygen species, as in the lungairways where its concentration is 140 times the normal level in bloodplasma. However, despite these actions of GSH, most workers in the fieldare of the opinion that they provide only a partial explanation for theresultant pathology.

In conclusion it is openly acknowledged by research workers that thetrue nature of the pathophysiology of CF is unknown and that the searchshould be extended for a missing factor or factors.

There is no single therapeutic procedure which successfully prevents theultimate progression of the pathologies which result in death. The majoreffort in the treatment of CF is devoted to combatingbroncho-pneumonitis. The objective is to minimise tissue damage byfacilitating bronchial clearance of viscous, infected mucus (chestphysiotherapy, broncho-dilators, mucolytics), and reducing bacterialburden (antibiotics).

Although the pathophysiology is not yet understood, viscous mucus in CFlungs is acknowledged as being a major factor in the pathogenesis of thelesions. Recently, it has been realized that DNA, actin and P.aeruginosa-derived alginate play a significant aggravating role inthickening secretions and exudates in CF. There is thus an ongoingstrategy to investigate the role of such substances in pathologicalsecretions in CF and thereby to develop agents based on this premise toalleviate some of the features of airway obstruction.

The first product to reach the marketplace as a result of this strategyis recombinant human DNAse (rhDNAse). It is well-established thatinfected secretions in CF lungs contain as much as 10% DNA as a resultof the continual breakdown of bacteria and neutrophils in chronicallyinfected lungs. Extracellular DNA forms long molecular chains whichincrease the visco-elasticity of the secretions. RhDNAse alters thephysical properties of CF secretions both in vitro and in vivo.Currently, some 50% of CF patients in the USA receive rhDNAse and it hasbeen demonstrated that patients cough less and have less thick sputum.Its impact on lung function, however, is small. Young patients with mildlung disease given a six months course of rhDNAse produced only a 3%benefit in FEVI, but it decreased the frequency of intravenousantibiotic courses by 34%.

Another compound believed to have the potential to decrease sputumviscosity is gelosin, which breaks down polymeric filamentous actin(F-actin), another component in the thick secretions which makes animportant contribution to its viscosity. Although polymeric F-actinmakes up around 10% of CF sputum, clinical studies on the efficacy ofgelosin have to date been disappointing.

With respect to patient tolerance of both agents, studies have shownthat gelosin and DNAse may activate inflammatory mediators such asInterleukin 8, and may explain the disappointing impact of thesecompounds on lung function.

A further new approach to thinning thickened secretion in CF patients isthe use of low-dose macrolide antibiotics, which interferes with theability of pseudomonads to manufacture alginate, a major component ofthe protective coating of these bacteria. When these bacteria reach acritical density, they produce large quantities of alginate, creating abiofilm which interferes with the penetrance of antibiotics, access ofacute inflammatory cells to the bacteria and hinders the clearance ofthe thickened secretions from airways in CF patients.

Separate products have therefore been individually developed with a viewto facilitating the clearance of mucus, DNA, actin and bacterial-derivedalginate in the grossly thickened secretions in CF patients with onlyrelative success.

In the knowledge that lamellar bodies are secreted by serous cells inthe airways, and in light of the fact that it has been shown byApplicant that lamellar body secretion in extrapulmonary sites isresponsible for lubrication of serous cavities throughout the animalkingdom, ex vivo investigation of the effect of synthetic lamellarbodies on mucus, DNA, actin and bacterial-derived alginate was carriedout. As demonstrated herein, the results confirmed that syntheticlamellar bodies significantly decrease the viscosity of mucus, polymericDNA, actin and alginate in in vitro studies.

Therefore synthetic lamellar bodies possess the ability, as shown invitro, to alter the viscosity, adhesiveness and fluidity of theindividual and combined components of the thickened secretion in CFpatients, in contrast to current therapeutic endeavours which have beendirected at formulating specific agents for each component involved inthe pathological process.

Accordingly, the inventor maintains that CF may be treated by exposureof the composite, pathologically thickened secretions to syntheticlamellar bodies. This will be achieved by restoration of adequate locallevels of lamellar bodies, sufficient for the diminution of viscosityand adhesiveness, while increasing the fluidity and clearance ofpathological secretions.

Furthermore, the inventor maintains that in CF a key factor in thepathogenesis of the disorder is relative failure of the lamellar bodysystem to secrete adequate volumes of phospholipid microbodies tobalance the physical properties of mucus and the pathological debrisproduced by intercurrent local inflammation (DNA, actin and alginate).Thus therapeutic effect is achieved by regular intermittent applicationof lamellar bodies to the affected tubal systems and surfaces, where thelubricating microbodies, as has been demonstrated in vitro, penetratewith ease the interstices of the gel-like structure of the thickenedsecretions.

Other Disease States

There are a number of other disease states in which linearmacromolecules are central to their pathogenesis and progression of thecondition in a manner outlined in detail in cystic fibrosis, wherelamellar bodies or a lamellar body composition could be used tobeneficial effect. These include respiratory disorders such asbronchitis and chronic bronchitis, asthma, bronchiectasis and chronicobstructive pulmonary disease. Additionally, disorders affecting thegastro-intestinal, hepato-biliary, pancreatic and reproductive tractsare subject to organismal and auto-immune induced thickened secretions.A variety of wounds affecting skin, such as thermal and radiation bums,chronic ulcers and de-gloving injuries with extensive loss of epidermis,dermis and full thickness skin, share a common pathological feature ofadhesive and viscous exudates. Likewise, these constitute conditionswhere lamellar bodies or a lamellar body composition, could be used tobeneficial effect.

The Skin: Lamellar Bodies in the Treatment of Thermal and RadiationBurns, in Chronic Ulcers and in Wounding Involving Extensive Loss ofEpidermis, Dermis and Full Thickness Skin

In burning injuries to skin, areas of loss of epidermis and dermis, downto full thickness skin, present a raw wound which is typically coveredby viscous, highly adhesive exudates consisting of DNA and actinreleased from the nuclei of host-derived dead epidermal and dermalcells, leucocytes and pus cells, together with DNA and actin releasedfrom dead colonizing bacteria. The viscosity and adhesiveness of theexudates in many instances are further increased with the onset ofsignificant infection by alginate-secreting organisms such asPseudomonas aeruginosa, a frequent and troublesome infection in burnsinjuries.

The viscous and adhesive properties of the burns exudates give rise toseveral key pathological effects, which seriously compromise woundhealing. The most damaging effect of the exudates is its tenaciousadherence to wound dressings of all types and textures. Each timedressings are removed, the wound surface is traumatized, resulting in anout-pouring of fresh fibrin. This not only causes the patient extremepain but also, this regularly repeated insult creates stratified layersof dense fibrin which seriously retard the healing process and arepathogenetically responsible for over-production of collagen andconsequent scarification and deformity.

The viscous and adhesive bums exudate also impedes and restricts theingress and movement of cells essential for the scavenging of debris,the phagocytosis of bacteria and cells involved in structural repair andre-epithelialization.

The viscous exudates, DNA and actin, provide sanctuary areas whichfoster infection, while colonization by organisms which actively secretebiofilm (alginate) then further augment the pathological effect ofviscosity and adhesiveness. This sequential process progressivelyshields the colonizing organisms from attack by phagocytic cells andseriously restricts the penetration of antibiotics into the constantlythickening exudate.

It should be noted that lamellar bodies are normally produced in humanskin by the keratinocytes. We have shown that in cell culture,replicating immature human keratinocytes vigorously secrete lamellarbodies. Although the scientific community has not yet considered therole of lamellar bodies in the promotion of wound healing, our detectionby electron microscopy of their presence in skin and peritoneal wounds,strongly indicates the appropriateness of topical administration oflamellar bodies to wounds, particularly in burns, where the pathologicalpresence of viscous and adhesive linear macromolecules seriouslycompromises healing and repair.

Chronic Skin Ulcers

A diverse range of chronic ulcers are encountered in skin. This occursmost commonly in lower limb and foot, particularly in diabetes mellitus,venous stasis and peripheral vascular disease. As with burns injuries,viscous and adhesive exudates consisting of DNA, actin andbacterial-derived alginates when present as a film on the surface of theulcer base can seriously impair wound healing. This is a major factor inthe maintenance of chronicity in ulcers of this type. As in the standardtreatment of burns, adhesion of occlusive dressings to the ulcer baseresults in continual micro-trauma to the delicate membrane each timethey are removed. This process therefore causes repeated episodic oozingof fresh fibrin over the base of the ulcer. Since fibrin is the majorstimulus to production of granulation tissue, frequent episodes offibrin exudation create a permanent state of early-stage wound repair inthe base of the ulcer, which effectively blocks the natural progressionto resolution and re-epithelialization.

Wounding Involving Extensive Denudation of Epidermis, Dermis Down toFull Thickness Skin (De-Gloving Injury)

Loss of skin in injuries of this nature creates a form of wound notdissimilar to that caused by burning. Likewise, occlusive dressingsadhere tenaciously to the range of viscous molecules present in thesurface exudate. Frequent or daily stripping of occlusive bandagesprovokes episodic exudation of fibrin, resulting in similar pathologicalsequelae encountered in burns and chronic ulcers.

Methods of Therapeutic Application

The following methods are applicable for diverse types of wounds, burns,chronic ulcers, de-gloving injuries. Wounds are sprayed with asuspension of lamellar bodies or a lamellar body composition before thedressings are applied. Preferably dressings should havemicro-perforations of a diameter sufficient to allow passage of lamellarbodies through into the underlying wound. Likewise, the density of thepores per unit area shall be appropriate to adequate delivery oflamellar bodies to the underlying interface between the dressing and thewound when a suspension of lamellar bodies are sprayed over the outersurface of the dressing. The perforations on the dressings preferablyshould be of a density which does not compromise the occlusiveproperties of the dressing with respect to ingress of bacteria. At anappropriate time interval before its removal, dependent on the rate ofdelivery of lamellar bodies to the wound surface, the dressing will besprayed with a suspension of lamellar bodies, which passes through theperforations into the space between it and the wound surface.

Dressings Incorporating Lamellar Bodies

A further method of application is the provision of dressingsincorporating lamellar bodies which are released gradually from theunder surface of the dressing onto the wound surface. This may beachieved by their containment in a thin envelope within the dressing,which acts as a reservoir for slow release of lamellar bodies throughmicroscopic perforations by incorporating a porous membrane on the innersurface.

Lamellar bodies may also be incorporated in a dressing whose undersurface is coated with a matrix or a gel from which they are released.Biocompatible gels would include hyaluronan and/or chondroitin sulphate.These gels are of lower viscosity compared to the pathological exudatespresent in wounds against which the lamellar bodies are directed, andthe presence of lamellar bodies would confer a fluid, non-stick surfaceat the interface between wound and dressing.

Use of Lamellar Bodies in Disorders of the Mucous Membranes (Mucositis)

Inflammation of mucous surfaces, particularly those close to the bodyopenings (buccal cavity, oropharynx, larynx, male and femalegenito-urinary tracts, and anal canal) are subject to painfulinflammation, erosion and ulceration. A generic term, mucositis, denotesa not uncommon affliction caused by a wide spectrum of etiologicalfactors, ranging from radiation, chemotherapeutic agents, drugs,auto-immunity, to allergies and infections. A characteristic feature ofthese conditions is the dryness of the surfaces, which become coveredwith sticky exudates consisting of viscous mucus, viscous DNA and actinfrom dead cells and bacteria. In extreme cases, opposing surfaces adhereone to the other forming synechiae, as in the oropharynx, prepuce, labiaand vagina. Swallowing or slight movements affecting the surfaces arevery painful. Separation of opposing surfaces causes repeatedmicro-trauma to the mucous membrane such as to promote ulceration andprevent healing. Specific dermatological disorders, which give rise tothese problems include Stevens-Johnson syndrome, Pemphigus, Behcet'sdisease, systemic lupus erythematosis and other bullous disorders. Asuspension of lamellar bodies in physiological saline applied by regularspraying of the affected areas, or the application of lamellarbody-containing gels or matrices will restore lubricity and non-stickproperties to the dry, adherent surfaces. Application of lamellar bodiesto affected mucous surfaces will decrease viscosity of the pathologicalsecretions and exudates, solubilized crusting and prevent formation ofsynechiae.

Use of Lamellar Bodies in inflammatory Conditions of the Eye

Lamellar bodies are found in tears. We have shown by immunocytochemistryusing the monoclonal antibody that the lacrimal epithelia stainpositively for surfactant protein A. This indicates that lamellar bodysecretion is a constitutive function of lacrimal glands in man. Themucous membrane of the eyelids and the conjunctivae are normallymoistened by a thin fluid. In pathological conditions the externalsurfaces of the eye may become dry and/or covered by thickened viscoussecretions or exudates. These exudates may form crusts whose abrasiveeffect further excoriates and traumatises the inflamed surfaces. A widevariety of disorders give rise to lesions of this nature. In the commonautoimmune disorders, rheumatoid and Sojgren's syndrome, an irritatingdryness of the eyes called kerato-conjunctivitis sicca is a frequentoccurrence. A wide variety of other disorders, inflammatory disordersdue to viral or bacterial infection, allergies, adverse drug reactionsgive rise to blepharitis and conjunctivitis with accompanying viscoussecretions and exudates. Regular administration of suspensions oflamellar bodies as eyedrops would be an appropriate therapeutic strategywhose usefulness and biocompatibility would be ascribed to therestorative effect of natural tears. Lamellar bodies may also be appliedin the form of gel, matrices or slow-release devices or excipients.

Production of Synthetic Lamellar Bodies

The focus of the present invention is the therapeutic use of lamellarbodies. The following information provides details of their productionand the key features and composition, which characterizes them as anovel microbody. In contrast to liposomes, whose invention from theoutset was not based on a concept in any way related to anaturally-occurring microbody, synthetic lamellar bodies are designed tomimic a range of biological activities ascribed to naturally-occurringlamellar bodies in tissues and body cavities. The principaldifferentiating characteristics of naturally-occurring lamellar bodiesare:

-   -   1) On contact, lamellar bodies, both divide easily and fuse        readily one with the other in body cavities and tissues when        subjected to movement and shear stress.    -   2) The provision of lubricity.    -   3) The automatic surface adsorption of planktonic molecular        debris arising from inflammatory processes, etc.    -   4) The automatic, non-receptor-mediated endocytosis of lamellar        bodies by the monocyte/phagocyte system, (and most especially by        antigen presenting cells, dendritic cells and macrophages).

These properties are crucially dependent on the nature of the mix of thedifferent species of phospholipids (phosphatidylcholine, sphingomyelin,phosphatidylethanolamine, phosphatidylserine and phosphatidylinosotol)and the nature of their fatty acid chains whose heterogenicity, lengthand range of saturation and unsaturation leading to lamellar fluidity isof key importance for their striking range of biological activities.

In contrast to lamellar bodies, liposomes must on no account split orfuse. This is achieved by their different composition, conferring anecessary rigidity through high proportions of cholesterol and use ofmore rigid phospholipids which, together greatly reduces membranefluidity. Liposomes are not designed to confer lubricity. Thecomposition of liposomes is chosen not to confer any surface adsorptiveproperties, a feature which would be totally incompatible with liposomalusages. Liposomes are specifically designed not to be phagocytosed bythe monocyte/phagocyte system, which would preclude any form oforgan-targeted drug delivery which has been their prime, relativelyunsuccessful use. Hence the development of Stealth liposomes in anattempt to obviate the biologically incompatible inherent paradox in thecurrent formulation of liposomes.

The principal phospholipid constituents of lamellar bodies arephosphatidylcholine (PC), sphingomyelin (SPH), phosphatidylethanolamine(PE), phosphatidylserine (PS), phosphatidylinositol (PI) andlysolecithin (LPC). The phospholipid composition of lamellar bodiesshows slight variation according to the cell of origin.

PC is the principal phospholipid in lamellar bodies, irrespective ofsite of origin. The percentage PC concentration varies from around 70%in lung lavage to 45% in synovial fluid (Refs). The next phospholipid inranking concentration is SPH (15-23%). Thereafter, PE, PS, PI, PG andLPC are present in varying, single digit percentage concentrations inlamellar bodies according to site of origin.

The preferred composition of phospholipids and cholesterol forphospholipid multilamellar microbodies comprises: PC 54%:SPH 19%:PE8%:PS 4%:PI 3%:cholesterol 10%. These values are median and thefollowing range of compositions have been found in natural lamellarbodies (private research): PC 44-70%, SPH 15-23%, PE 6-10%, PS 2-6%, PI2-4%, Cholesterol 4-12%. These figures are percentage by weight.

LPC may also be incorporated into the multilamellar microbodies at 2% byweight which follows the range found in natural lamellar bodies of 0-3%.

Liposomes are made by those skilled in the art with high cholesterolconcentrations to improve their rigidity. Liposomes containingcholesterol at 20% or below would be considered to be cholesterol poor.Thus liposomes incorporating a high ratio (50%) of cholesterol, where itis equimolar with the phospholipids, have a highly stable structure andare the pivotal concept for the creation of liposomes. Until thisinvention, it would not to our knowledge have been obvious to try usinglow-cholesterol multilamellar microbodies since the late discovery oflamellar bodies as a ubiquitous biological phenomenon remains unknownand unquoted by anyone working in the liposomal field. The cholesterolcontent of lamellar bodies derived from pulmonary alveoli has been foundto contain around 10% cholesterol.

The presence of sphingomyelin in natural lamellar bodies and in thesynthetic lamellar bodies claimed in the present invention is important.Sphingomyelin is not generally used in liposomes and serves to giveflexibility and softness to lamellar bodies. Conventional liposometechnology teaches that rigidity is mandatory for the delivery ofchemicals; however, we have found that flexible, low-cholesterol,sphingomyelin containing synthetic lamellar bodies are ideal fordelivery of antigen to antigen presenting cells and, as demonstrated byvigorous non-receptor mediated endocytosis by dendritic cells ofsynthetic lamellar bodies, first described by the inventor.

Phospholipid multi-lamellar microbodies (synthetic lamellar bodies) areprepared by a technique similar to that used to produce hand-shakenmulti-lamellar vesicles. The phospholipid mixture, together withcholesterol in the percentages given by weight, is dissolved in achloroform/methanol solvent mixture (2:1 vol/vol). The lipid solution isintroduced into a round-bottomed flask and attached to a rotaryevaporator. The flask is evacuated and rotated at 60 r.p.m. in athermostatically controlled waterbath at a temperature of 30° C. until adry lipid film is deposited. Nitrogen is introduced into the flask andthe residual solvent is removed before its connection to a lyophilizerwhere it is subjected to a high vacuum at room temperature for one hour.After release of the vacuum and following flushing with nitrogen, salinecontaining solutes (selected antigen) for entrapment is added. The lipidis hydrated within the flask, flushed with nitrogen, attached to theevaporator, and rotated at 60 r.p.m. at room temperature for thirtyminutes. The suspension is allowed to stand for two hours at roomtemperature to complete the swelling process.

It should be noted that the embodiments disclosed above are merelyexemplary of the invention, which may be embodied in many differentforms. Therefore, details disclosed herein are not to be interpreted aslimiting, but merely as a basis for Claims and for teaching one skilledin the art as to the various uses of the present invention in anyappropriate manner.

Pharmaceutical Compositions and Methods of Delivery

Another aspect of the invention provides for pharmaceutical compositionscomprising purified lamellar bodies for the modification of linearmacromolecules. These lamellar bodies may be synthetic or naturallyoccurring and may act as surrogate lamellar bodies in body cavities,blood vessels, ducts, sinuses and tissues to modify mucus viscosity fortherapeutic purposes.

One embodiment features treatment of a wide range of diseases andconditions with pharmaceutical compositions containing synthetic ornaturally occurring lamellar bodies or microbodies and acceptablecarriers and excipients. Moreover, a further embodiment may include apharmaceutical composition designed for use in local treatment ofconditions having a preponderance of heavy mucous secretions. Anotherembodiment may include a pharmaceutical composition designed forsystemic use alone or with other standard treatment modalities known tothose skilled in the art.

Such compositions comprise a therapeutically effective amount of anagent, and a pharmaceutically acceptable carrier. In one embodiment, thepharmaceutical composition may be presented in unit dosage forms tofacilitate accurate dosing. The term “unit dosage forms” refers tophysically discrete units suitable as unitary dosages for human subjectsand other mammals, each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect, inassociation with a suitable pharmaceutical excipient. Typical unitdosage forms include prefilled, premeasured ampules or syringes of theliquid compositions or pills, tablets, capsules or the like in the caseof solid compositions. The unit can be, for example, a single use vial,a pre-filled syringe, a single transdermal patch and the like. The unitdosage form can be in unit dose or unit-of-use packages. As is known tothose skilled in the art, a unit dose package is a convenient, patientready unit. An exemplary unit dosage form of the present invention wouldbe a 5 ml suspension of lamellar bodies in which there would be 54 mg ofphosphatidylcholine, 19 mg of sphingomyelin, 8 mgphosphatidylethanolamine, 4 mg phosphatidylserine, 3 mgphosphatidylinosotol and 10 mg cholesterol. In a particular embodiment,the term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. The term “carrier” refers to adiluent, adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Sterile isotonic aqueous buffer is a preferred carrierwhen the pharmaceutical composition is administered intravenously.Saline solutions and aqueous dextrose and glycerol solutions can also beemployed as liquid carriers, particularly for injectable solutions.Suitable pharmaceutical excipients include starch, glucose, lactose,sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate,glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol,propylene, glycol, water, ethanol and the like. The composition, ifdesired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the subject. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lidocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The amount of the compound of the invention which will be effective inthe treatment of the conditions described herein can be determined bystandard clinical techniques based on the present description. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each subject's circumstances.However, suitable dosage ranges for intravenous administration aregenerally about 20-500 micrograms of active compound per kilogram bodyweight. Suitable dosage ranges for intranasal administration aregenerally about 0.01 pg/kg body weight to 1 mg/kg body weight. Effectivedoses may be extrapolated from dose-response curves derived from invitro or animal model test systems.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects (a)approval by the agency of manufacture, use or sale for humanadministration, (b) directions for use, or both.

In a specific embodiment, it may be desirable to administer thepharmaceutical compositions of the invention locally to the area in needof treatment; this may be achieved, for example, and not by way oflimitation, by local infusion during surgery or by spraying the solutioncontaining the lamellar bodies onto the exposed tissue followingsurgery, by topical application, by injection, by means of a catheter,or by means of an implant, said implant being of a porous, non-porous,or gelatinous material, including membranes, such as sialasticmembranes, or fibers or co-polymers such as Elvax (see Ruan et al, 1992,Proc Natl Acad Sci USA, 89:10872-10876). In one embodiment,administration can be by direct injection by aerosol inhaler.

In yet another embodiment, the lamellar bodies can be delivered in acontrolled release system. In one embodiment, a pump may be used (seeLanger, supra; Sefton (1987) CRC Crit. Ref. Biomed. Eng. 14:201;Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J.Med. 321:574). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J. (1983) Macromol. Sci. Rev. Macromol.Chem. 23:61; see also Levy et al. (1985) Science 228:190; During et al.(1989) Ann. Neurol. 25:351; Howard et al. (1989) J. Neurosurg. 71:105).In yet another embodiment, a controlled release system can be placed inproximity of the therapeutic target, thus requiring only a fraction ofthe systemic dose (see, e.g., Goodson, in Medical Applications ofControlled Release (1984) supra, vol. 2, pp. 115-138). Other suitablecontrolled release systems are discussed in the review by Langer (1990)Science 249:1527-1533.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the compositions described herein for therapeuticapplications, and are not intended to limit the scope of what theinventors regard as their invention. Efforts have been made to ensureaccuracy with respect to numbers used (e.g., amounts, temperature, etc.)but some experimental errors and deviations should be accounted for.Unless indicated otherwise, parts are parts by weight, molecular weightis average molecular weight, temperature is in degrees Centigrade, andpressure is at or near atmospheric.

Example 1 Ex Vivo Investigation of Effect of Synthetic Lamellar Bodieson Mucus, DNA, Actin and Alginate Gels and Ear Wax

Basic laboratory bench tests have been performed on the effect on thephysical properties of linear macromolecules of the type involved in thepathophysiology of secretions and exudates when mixed with phospholipidmicrobodies (synthetic lamellar bodies).

Materials

The materials tested were chosen in the knowledge of the probablemolecular status of naturally-occurring polymers in physiologic and inpathologic conditions. The range of materials commercially available forselection was however, restricted. Nevertheless, our accumulatedexperience in this field directed that the main thrust of theexperiments on these gel-forming polymers would be focused ondemonstrating a molecular species interaction between microbodies andpolymers. In this regard it was a basic premise of the experiments thatthe behaviour of the gels was predictably similar, because of a sharedthree-dimensional structure of this class of substances. Thus theselection of mucus, DNA, actin and alginate preparations were matched tothe probable physical state of the polymers in vivo.

Preparation of the gels, their range of concentration and hydration wascalculated to mimic as closely as possible the putative rangeencompassing normal and pathological conditions in humans.

Characteristics of Experimental Mucus Tested

The mucus tested was derived from bovine, submaxillary glands assupplied by Sigma (A70030). This is one of the few commerciallyavailable products whose composition is characterized. This particularagent has been tried and tested in investigation of the physicalcharacteristics of the gel status of mucus in research on its viscosityin inflammatory respiratory disorders, including CF.

In normal secretions, e.g. upper respiratory tract surface fluid, mucusis present at low concentrations of between 0.5% and 1% by weight. 99%of the secretions consist of water containing electrolytes. Mucus has anextended polypeptide backbone (apmucin) with numerous oligosaccharideside-chains. Covered by abundant polyanionic charges, mucus, onexocytotic release from goblet cells, becomes extensively hydrated.Mucus then forms a gel whose three-dimensional structure comprises atangled network of linear mucin polymers, devoid of intermolecularcross-bridges. In certain situations of hypersecretion and inflammation,the mucin concentration can rise to 3%-5% by weight.

Mucus has low solubility. In bench-testing of the physical properties ofthe bovine submaxillary mucus, solutions up to a concentration of 5%were possible using saline. For testing with lamellar bodies,concentrations at 2.5% and 3.5% were employed to mimic those encounteredin pathological situations. Saline (0.9%) was used as the diluent forall mucous samples tested.

Mucin monomers are polymerized through end-to-end disulphide bonds toform linear polymers which, in respiratory mucus, are at least 30μ long.These dimensions should be viewed in context of the size of lamellarbodies which range between 0.5μ-2.0μ. Thus lamellar bodies are of adimension which can easily pass through a tangled network of mucous“fibers” as observed by light microscopy.

Characteristics Of Experimental DNA Tested

The DNA chosen was polymeric in form and derived from salmon testes(Sigma D1626). Like mucus, the DNA preparation selected consists oflinear polymers which form a tangled “fibrous” gel. It is poorly solublein water and concentrations of over 5% by weight produce a very stiffgel. In the tests carried out with lamellar bodies, concentrations of 1%were regularly employed, and the solvent used was always 0.9% saline.This concentration of 1% was selected as probably being representativeof leucocyte (pus cells) and organismal-derived DNA present in thickpathological secretions in various disorders, ranging from CF through tochronic otitis media.

A 1% solution of polymeric DNA in physiological saline is a clear,syrupy liquid whose gel state possesses high viscosity and adhesiveness.

Characteristics of Experimental Combined Mucus and DNA Gels

Solutions of 2.5% mucus in 0.9% saline and 1% DNA in 0.9% saline weremade up separately. They were then carefully mixed and stirred, avoidingthe creation of bubbles. The gels mixed well, without producing anychanges in turbidity. It is noteworthy that in published studies,samples from patients of thick, viscous, infected respiratory mucus whenstained for DNA and mucus, showed in wet preparations examined byconfocal microscopy that the different polymers were intimately mixed inthe three-dimensional structure of the gel. We were therefore confidentthat the samples of combined mucus and DNA gel prepared for testing withlamellar bodies were most probably representative of viscous effusionsin pathological situations in a variety of in vivo disorders, rangingfrom CF through to chronic otitis media.

Characteristics of Synthetic Lamellar Bodies

The lamellar bodies tested were produced by the mixing of two organicsolvents containing the phospholipids and cholesterol components. Basedon analysis of lamellar bodies produced by human mesothelial cells, sixconstituents (five phospholipids and cholesterol) are used to makemicrobodies consisting of tightly-packed phospholipid bilayers with anultrastructural geometry and periodicity (distance between bilayers)confirmed by electron microscopy to be identical to that ofnaturally—occurring lamellar bodies. The range of size of the syntheticlamellar bodies (0.2-3.5 μm) was closely similar to that found intissues and body cavities in man.

The lamellar bodies are made up in a physiological saline to a standardsolution containing 10×10⁹ microbodies per ml. The lamellar body densityestimates are checked by turbidity comparison methods, while sizedistribution is obtained using flow cytometry.

Methods

Considering the widespread occurrence of mucus throughout the body, theliterature on experimental work on its molecular biology is surprisinglyscant. Current literature on the pathogenesis of cystic fibrosis admitsignorance of the basic pathogenetic mechanisms pertaining to mucus indifferent disorders. It is agreed by those in this field thatinvestigation of visco-elastic properties of biological fluids and gelsposes many technical and instrumental difficulties.

Applied viscometry has been developed exclusively for industrialpurposes, and all instruments are designed for large fluid volumes. Purebiological polymers are extremely expensive and measurement of multiplesamples, where minimal testing volumes per sample on availableinstruments are 10-20 ml, can be prohibitive.

Standard industrial viscometers employ mechanical techniques, inducingshears, causing alignment of linear polymers and thus disentangling thegel. Therefore, measurement of biological gels in standard viscometers(e.g. rotating disc and cone) can significantly alter the viscosity ofthe sample during the test. On the other hand, rheology of biologicalfluids has evolved using instruments for investigation of blood which isa non-Newtonian fluid. Likewise plasma viscosity is highly dependent onglobular, not linear, proteins like albumin at 45 g/l and globulin at 35g/l. Only one company (recently disbanded) has designed instruments formeasurement of viscosity in biological fluids.

To obviate the difficulties in using instruments not modified fortesting viscosity in biological samples, a series of pilot studies werecarried out employing simple devices to identify the most suitablemethod for providing reliable measurements of change in viscosity ingels mixed in saline with and without added lamellar bodies.

A method was developed which measured distance travelled in unit time byfixed volumes of test and control samples, down a fixed incline undergravity (Galileo technique), where the density and water content of thesamples were comparable, and the surface chosen was of homogeneouscomposition, surface texture and charge. The most appropriate slopetested was the surface of a plastic Petri dish.

All laboratory ware used (i.e. containers, pipettes, etc) were ofplastics which did not contain plasticisers of the type DEHP, sincelamellar bodies leach out and are altered by this plasticiser.

Gels were freshly made up to the appropriate concentrations withadequate and careful mixing, ensuring complete solubility and hydrationof the polymers.

A measured volume of 0.9% saline containing lamellar bodies was thenadded to test samples and an identical volume of 0.9% saline only wasadded to the control samples. Following careful, but complete mixing,samples were incubated at 37° C. for 1 hour.

After incubation and further careful mixing, droplets (ranging from 30μl-100 μl) were accurately pipetted on a start-line marked on the backof the Petri dish, which also had a parallel “finish” line drawn at adistance of 5 cms. As the Petri dish was elevated to an angle of 90°, anelectronic stopwatch was triggered which was used to record byobservation the time taken for the droplet to stream down to the 5 cmmarker.

Results

The key elements of the findings are summarized in Tables 1-3, describedbelow.

Table 1. Effect of varying concentrations of synthetic lamellar bodieson mucus fluidity compared with use of saline diluent as control. Eachsample tested was exactly 30 μl. The angle of incline was 90°.

Table 2. Effect at 37° C. of varying concentrations of syntheticlamellar bodies on DNA fluidity compared with use of saline diluent ascontrol. Each sample tested was 100 μl. The angle of incline was 90°.

Table 3. Effect at 37° C. of synthetic lamellar bodies on fluidity of acombined mucus—DNA gel at a ratio of one third volume synthetic lamellarbodies to two thirds DNA & mucus gel compared with control using onethird volume of 0.9% saline. Each sample tested was 40 μl. The angle ofincline was 90°.

Effect of Varying Concentrations of Lamellar Bodies on Mucus Gels

The experiment most representative of the alterations observed in theviscosity of mucus was the one illustrated in Table 1 in which 3.5%mucus in saline was sequentially diluted with increasing volumes of 0.9%saline (controls) and 0.9% saline containing 10×10⁹ lamellar bodies. Theratios of gel to diluent tested for both test and control samples were19:1, 9:1, 8:2, 7:3, 6:4.

No movement was observed in any of the samples in which the diluentmixed with the mucus gel was physiological saline only, in contrast tothe test samples in which the saline diluent contained lamellar bodies,where even at a ratio of 19:1 there was significant movement in thesolution down the inclined plane. Thereafter, the speed of travelincreased steeply and at the ratio of nine parts mucus to one partlamellar body solution, the fluid travelled five centimeters in sixseconds. At ratios of seven parts to three, a maximum speed of travelwas reached at five centimeters per second. At these concentrations thetests demonstrated highly significant differences in the behaviorbetween the mucus gels mixed with matched volumes of saline alone,compared with test samples where the saline contained lamellar bodies.These results tested within the stated range and concentrationsdemonstrate that the presence of the phospholipid microbodies confer aremarkable lubricity to the gel, independent of the fluid content of thediluent.

Effect of Varying Concentrations of Lamellar Bodies on DNA Gels

The experiment most representative of the alterations observed in theviscosity of DNA was the one illustrated in Table 2, one in which 1% DNAin saline was sequentially diluted with increasing volumes of 0.9%saline (controls) and 0.9% saline containing 10×10⁹ lamellar bodies. Theratios of gel to diluent tested for both test and control samples were19:1, 9:1, 8:2, 7:3, 6:4, 5:5. Minimal movement was observed in bothtest and control at dilutions from 19:1 down to 8:2. Thereafter, testsamples in which the diluent contained lamellar bodies in ratios of 7:3,6:4, 5:5 exhibited a steep increase in speed of travel to 2.5 cms persecond, as compared to controls where the maximum speed was 5 cms in 4min 20 sec.

Effect of Varying Concentrations of Lamellar Bodies on Combined DNA &Mucus Gels

The experiment most representative of the alterations observed in theviscosity of combined DNA and mucus gels was the one illustrated inTable 3. The combined gel consisted of 333 μl of 2.5% mucus in 0.9%saline, together with 333 μl of 1% DNA in 0.9% saline. In the controlsample 333 μl of 0.9% saline was added as diluent, whereas in the testsample 333 μl of saline containing 10×10⁹ lamellar bodies was added tothe combined gel. No movement was obtained in the control sample,whereas the test sample containing lamellar bodies travelled 5 cms in 2sec at a speed of 2.5 cms per sec. Therefore at a ratio of one thirddiluent containing lamellar bodies added to two thirds combined mucusand DNA gel, there was complete contrast between the fluidity of thecontrol and test sample.

Alginate and Actin Gels

Broadly similar results as those found in the testing of the effect oflamellar bodies on mucus and DNA were obtained in comparing their effecton alginate and actin gels. The agents tested were muscle-derived,porcine actin G (Sigma A0541) made up as a 0.5% solution in 0.9% saline.Alginic acid, a polyuronic acid composed primarily of anhydro-beta-Dmannuronic acid residues with 1-4 linkage (Sigma A70030) was made up asa 2.5% solution in 0.9% saline. Light microscopic observations showedintimate mixing of lamellar bodies between the “fibers” of the gels.

Effect of Lamellar Bodies on Mucus Plugs Blocking Tympanostomy Tubes(Vent Tubes)

In vitro laboratory experiments were carried out in which vent tubeswere filled with 5% mucus gel and allowed to dry overnight. The tubeswere inserted in the spigot of a specially chosen 2 ml syringe, whoseconical profile accommodated precisely the outer diameter of the venttube, to achieve complete occlusion. 0.5 ml suspension of lamellarbodies was pipetted into the barrel of the syringe and its effectobserved. Vent tubes were unplugged at room temperature in a period thatranged from 30-60 minutes. Saline controls had no effect after 6 hours.

Effect of Lamellar Bodies on Ear Wax

Human ear wax is a mixture of substances. The two main constituents aredesquamated squamous cells and cerumen. The squamous epithelium whichlines the external auditory canal occupies an unusual position for skin.Over the rest of the body the epidermis is renewed by constant loss ofsurface cells and keratin by abrasion. Loss by abrasion is not possiblein the auditory canal. To overcome this difficulty the surfaceepithelium continually migrates centrifugally from the eardrum outwards.Cerumen is a waxy secreted by modified sweat glands. These are situatedin the sub-epidermal tissue from where they discharge their secretionthrough ducts onto the surface of the external auditory canal.

We have carried out tests in the laboratory on the effect of lamellarbodies on samples of wax obtained from patients, and have shown that thewax disintegrates over a period ranging from 12-24 hours. Microscopicobservations on the process show penetration between the plates ofsquames by lamellar bodies, lubricating and separating the adhesivelayers of dead cells which led to disintegration of the wax.

Conclusions

The mixing of a saline solution containing lamellar bodies with a mucusgel results in significant reduction in the adhesiveness and viscosityof the mixture, compared to a control mucus gel which has been mixedwith an equal volume of saline alone, when measured by rate of movementdown an inclined plane of identical composition, surface texture andcharge. Similar results were obtained when lamellar bodies were added toDNA, actin and alginate gels, and also to combined mucus and DNA gels.Significant changes in speed of flow, and hence fluidity, wereencountered when the ratio of the volume of standard solution containinglamellar bodies added to the gel was around 3 to 7.

TABLE 1 Effect of varying concentrations of synthetic lamellar bodies onmucus fluidity compared with use of saline diluent as control. Eachsample tested was exactly 30 μl. The ingle of incline was 90°. CONTROLTEST 3.5% Mucus in 0.9% saline:saline diluent 0.9% saline 3.5% Mucus in0.9% saline:Synthetic lamellar bodies in 0.9% saline Volume&Concentration Dilution Time to Travel 5 cms Dilution Volume&Concentration 950 μl mucus + 50 μl NaCl 19:1  No movement 3 m 50 sec19:1  950 μl mucus + 50 μl LMS @ 5 min 900 μl mucus + 100 μl NaCl 9:1 Nomovement 6 sec 9:1 900 μl mucus + 100 μl LMS @ 5 min 800 μl mucus + 200μl NaCl 8:2 No movement 2 sec 8:2 800 μl mucus + 200 μl LMS @ 5 min 700μl mucus + 300 μl NaCl 7:3 No movement 1 sec 7:3 700 μl mucus + 300 μlLMS @ 5 min 600 μl mucus + 400 μl NaCl 6:4 No movement 1 sec 6:4 600 μlmucus + 400 μl LMS @ 5 min

TABLE 2 Effect at 37° C. of varying concentrations of synthetic lamellarbodies on DNA fluidity compared with use of saline diluent as control.Each sample tested was 100 μl. The angle of incline was 90°. CONTROLTEST 1% DNA in 0.9% saline:diluent 0.9% saline 1% DNA IN 0.9%saline:Synthetic lamellar bodies in 0.9% saline Volume &ConcentrationDilution Time to Travel 5 cms Dilution Volume &Concentration 950 μlDNA + 50 μl NaCl 19:1  10 mm @ 5 min 11 mm @ 5 min 19:1  950 μl DNA + 50μl LMS 900 μl DNA + 100 μl NaCl 9:1 10 mm @ 5 min 11 mm @ 5 min 9:1 900μl DNA + 100 μl LMS 800 μl DNA + 200 μl NaCl 8:2 25 mm @ 5 min 31 mm @ 5min 8:2 800 μl DNA + 200 μl LMS 700 μl DNA + 300 μl NaCl 7:3 45 mm @ 5min 22 sec 7:3 700 μl DNA + 300 μl LMS 600 μl DNA + 400 μl NaCl 6:4 4min 20 sec  2 sec 6:4 600 μl DNA + 400 μl LMS 500 μl DNA + 500 μl NaCl5:5 2 min 32 sec  2 sec 5:5 500 μl DNA + 500 μl LMS

TABLE 3 Effect at 37° C. of synthetic lamellar bodies on fluidity of acombined mucus - DNA gel at a ratio of one third volume syntheticlamellar bodies to two thirds DNA &mucus gel compared with control usingone third volume of 0.9% saline. Each sample tested was 40 μl. The angleof incline was 90°. CONTROL TIME TO TRAVEL 5 CMS TEST 333 μl 2.5% mucusin 0.9% saline No movement @ 5 min 2 sec 333 μl 2.5% mucus in 0.9%saline 333 μl 1% DNA in 0.9% saline 333 μl 1% DNA in 0.9% saline 333 μl0.9% saline as diluent 333 μl LMS in 0.9% saline

1. (canceled)
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 10. (canceled) 11.(canceled)
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 13. (canceled)
 14. (canceled)
 15. (canceled)16. A method of treating Otitis Media comprising the steps of: a)inserting a needle through the tympanic membrane; b) introducing acomposition including lamellar bodies through the needle into the ear;and c) allowing the lamellar bodies to modify the viscosity of the mucinin the ear such that it is capable of draining from the ear.
 17. Themethod of claim 16, wherein the composition is introduced into themiddle ear.
 18. The method of treating Otitis Media as set forth inclaim 16 or 17, comprising the steps of: a) inserting a needle throughthe tympanic membrane; b) introducing a composition including lamellarbodies through the needle into the ear, and c) allowing the lamellarbodies to modify the composition and the biological properties of thecontents present in the pathological secretion, including linearmacromolecules of the type mucus, DNA, actin and alginate, to the effectthat the physical properties of the secretions such as viscosity andadhesiveness are altered, permitting therapeutic drainage of the middleear.
 19. The method of treating Otitis Media as set forth in claim 16 or17, comprising the steps of: a) inserting a catheter into the pharyngealopening of the Eustachian tube; and b) introducing a composition,including lamellar bodies through the catheter into the ear.
 20. Amethod of performing a functional endoscopic sinus surgery (FESS)procedure on the sinus of a patient comprising the steps of: a) applyinga composition including lamellar bodies to the sinus; b) allowinglamellar bodies in the composition to modify the viscosity of the linearmacromolecules in the area of the sinus such that it is capable of beingremoved; and c) introducing a surgical instrument into the nasal passagesuch as to remove tissue from the sinus.
 21. The method of claim 20,wherein said linear macromolecules are selected from the groupconsisting of mucus, DNA, actin and alginate.
 22. A method of treating amammalian subject suffering from at least one condition selected fromthe group consisting of sinusitis, disorders of the alimentary andgenito-urinary tracts, ducts and surfaces of body cavities filled orcovered by thickened secretions or extrudates during surgicalprocedures, inflammatory disorders of the respiratory tract, cysticfibrosis, bronchitis, and nasal congestion comprising the step of:providing to said subject a therapeutically effective amount of lamellarbodies wherein said lamellar bodies comprise phosphatidylcholine,sphingomyelin, phosphatidyl ethanolamine, phosphatidyl serine,phosphatidyl inositol and cholesterol.
 23. The method of claim 22wherein the lamellar bodies comprise about 44-70% phosphatidylcholine,about 15-23% sphingomyelin, about 6-10% phosphatidyl ethanolamine, about2-6% phosphatidyl serine, about 2-4% phosphatidyl inositol and about4-12% cholesterol by weight.
 24. The method of claim 22 wherein thelamellar bodies comprise about 54% phosphatidylcholine, about 19%sphingomyelin, about 8% phosphatidyl ethanolamine, about 4% phosphatidylserine, about 3% phosphatidyl inositol and about 10% cholesterol byweight.
 25. The method of claim 23 or claim 24 wherein the lamellarbodies further comprise about 0-3% by weight of lysophosphatidylcholine.
 26. The method of claim 23 or claim 24 wherein the lamellarbodies further comprise about 2% by weight of lysophosphotidyl choline.27. The method of claim 22 wherein the at least one condition ischaracterized by problematic linear macromolecules wherein said linearmacromolecules are selected from the group consisting of DNA, mucin,actin, and bacterial- derived alginate.
 28. A method for increasing thefluidity of a subject's surface airway surface fluid and restoringmucociliary clearance of secretions of the respiratory tract comprisingapplying a composition including lamellar bodies, wherein said lamellarbodies comprise phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, phosphatidyl serine, phosphatidyl inositol andcholesterol, to a subject's respiratory passages to modify the physicalproperties of linear macromolecules of the type mucous DNA, actin andalginate in pathologically altered secretions.
 29. The method of claim28 wherein the composition contains suspended lamellar bodies whereinthe method includes the step of administering the suspended lamellarbodies into the respiratory passages as an aerosol spray.
 30. The methodof claim 29 wherein the suspended lamellar bodies are subjected toultrasonification during the step of administering inhaled aerosol.